1. Field of the Invention
Nucleic acid hybridization has been employed for investigating the identity and establishing the presence of nucleic acids. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. The availability of radioactive nucleoside triphosphates of high specific activity and the 32p labelling of DNA with T4 polynucleotide kinase has made it possible to identify, isolate, and characterize various nucleic acid sequences of biological interest. Nucleic acid hybridization has great potential in diagnosing disease states associated with unique nucleic acid sequences. These unique nucleic acid sequences may result from genetic or environmental change in DNA by insertions, deletions, point mutations, or by acquiring foreign DNA or RNA by means of infection by bacteria, molds, fungi, and viruses. Nucleic acid hybridization has, until now, been employed primarily in academic and industrial molecular biology laboratories. The application of nucleic acid hybridization as a diagnostic tool in clinical medicine is limited because of the frequently very low concentrations of disease related DNA or RNA present in a patient""s body fluid and the unavailability of a sufficiently sensitive method of nucleic acid hybridization analysis.
Current methods for detecting specific nucleic acid sequences generally involve immobilization of the target nucleic acid on a solid support such as nitrocellulose paper, cellulose paper, diazotized paper, or a nylon membrane. After the target nucleic acid is fixed on the support, the support is contacted with a suitably labelled probe nucleic acid for about two to forty-eight hours. After the above time period, the solid support is washed several times at a controlled temperature to remove unhybridized probe. The support is then dried and the hybridized material is detected by autoradiography or by spectrometric methods.
When very low concentrations must be detected, the current methods are slow and labor intensive, and nonisotopic labels that are less readily detected than radiolabels are frequently not suitable. A method for increasing the sensitivity to permit the use of simple, rapid, nonisotopic, homogeneous or heterogeneous methods for detecting nucleic acid sequences is therefore desirable.
Recently, a method for the enzymatic amplification of specific segments of DNA known as the polymerase chain reaction (PCR) method has been described. This in vitro amplification procedure uses two or more different oligonucleotide primers for different strands of the target nucleic acid and is based on repeated cycles of denaturation, oligonucleotide primer annealing, and primer extension by thermophilic polymerase, resulting in the exponential increase in copies of the region flanked by the primers. The different PCR primers, which anneal to opposite strands of the DNA, are positioned so that the polymerase catalyzed extension product of one primer can serve as a template strand for the other primer, leading to the accumulation of discrete fragments whose length is defined by the distance between the 5xe2x80x2-ends of the oligonucleotide primers.
Other methods for amplifying nucleic acids are single primer amplification, ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA) and the Q-beta-replicase method. Regardless of the amplification used, the amplified product must be detected.
Depending on which of the above amplification methods are employed, the methods generally employ from seven to twelve or more reagents. Furthermore, the above methods provide for exponential amplification of a target or a reporter oligonucleotide. Accordingly, it is necessary to rigorously avoid contamination of assay solutions by the amplified products to avoid false positives. Some of the above methods require expensive thermal cycling instrumentation and additional reagents and sample handling steps are needed for detection of the amplified product.
Most assay methods that do not incorporate amplification of a target DNA avoid the problem of contamination, but they are not adequately sensitive or simple. Some of the methods involve some type of size discrimination such as electrophoresis, which adds to the complexity of the methods.
One method for detecting nucleic acids is to employ nucleic acid probes. One method utilizing such probes is described in U.S. Pat. No. 4,868,104, the disclosure of which is incorporated herein by reference. A nucleic acid probe may be, or may be capable of being, labeled with a reporter group or may be, or may be capable of becoming, bound to a support.
Detection of signal depends upon the nature of the label or reporter group. If the label or reporter group is an enzyme, additional members of the signal producing system include enzyme substrates and so forth. The product of the enzyme reaction is preferably a luminescent product, or a fluorescent or non-fluorescent dye, any of which can be detected spectrophotometrically, or a product that can be detected by other spectrometric or electrometric means. If the label is a fluorescent molecule, the medium can be irradiated and the fluorescence determined. Where the label is a radioactive group, the medium can be counted to determine the radioactive count.
It is desirable to have a sensitive, simple method for detecting nucleic acids. The method should minimize the number and complexity of steps and reagents. The need for sterilization and other steps needed to prevent contamination of assay mixtures should be avoided.
2. Description of the Related Art
Methods for detecting nucleic acid sequences are discussed by Duck, et al., in U.S. Pat. No. 5,011,769 and corresponding International Patent Application WO 89/10415. A method of cleaving a nucleic acid molecule is disclosed in European Patent Application 0 601 834 A1 (Dahlberg, et al.).
Holland, et al., Clinical Chemistry (1992) 38:462-463, describe detection of specific polymerase chain reaction product by utilizing the 5xe2x80x2 to 3xe2x80x2 exonuclease activity of Thermus aguaticus DNA polymerase. Longley, et al., Nucleic Acids Research (1990) 18:7317-7322, discuss characterization of the 5xe2x80x2 to 3xe2x80x2 exonuclease associated with Thermus aguaticus DNA polymerase. Lyamichev, et al., Science (1993) 260:778-783, disclose structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases.
A process for amplifying, detecting and/or cloning nucleic acid sequences is disclosed in U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 and 5,008,182. Sequence polymerization by polymerase chain reaction is described by Saiki, et al., (1986) Science, 230: 1350-1354. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase is described by Saiki, et al., Science (1988) 239:487.
U.S. patent applications Ser. Nos. 07/299,282 and 07/399,795, filed Jan. 19, 1989, and Aug. 29, 1989, respectively, describe nucleic acid amplification using a single polynucleotide primer. The disclosures of these applications are incorporated herein by reference including the references listed in the sections entitled xe2x80x9cDescription of the Related Art.xe2x80x9d
Other methods of achieving the result of a nucleic acid amplification are described by Van Brunt in Bio/Technology (1990) 8(No.4): 291-294. These methods include ligase chain reaction (LCR), nucleic acid sequence based amplification (NASBA) and Q-beta-replicase amplification of RNA. LCR is also discussed in European Patent Applications Nos. 439,182 (Backman I) and 473,155 (Backman II).
NASBA is a promoter-directed, isothermal enzymatic process that induces in vitro continuous, homogeneous and isothermal amplification of specific nucleic acid.
Q-beta-replicase relies on the ability of Q-beta-replicase to amplify its RNA substrate exponentially under isothermal conditions.
Another method for conducting an amplification of nucleic acids is referred to as strand displacement amplification (SDA). SDA is an isothermal, in vitro DNA amplification technique based on the ability of a restriction enzyme to nick the unmodified strand of a hemiphosphorothioate form of its restriction site and the ability of a DNA polymerase to initiate replication at the nick and displace the downstream nontemplate strand intact. Primers containing the recognition sites for the nicking restriction enzyme drive the exponential amplification.
Another amplification procedure for amplifying nucleic acids is known as 3SR, which is an RNA specific target method whereby RNA is amplified in an isothermal process combining promoter directed RNA polymerase, reverse transcriptase and RNase H with target RNA.
One aspect of the present invention is a method for modifying an oligonucleotide. The method comprises incubating the oligonucleotide with a polynucleotide and a 5xe2x80x2-nuclease wherein at least a portion of the oligonucleotide is reversibly hybridized to the polynucleotide under isothermal conditions. The oligonucleotide is cleaved to provide (i) a first fragment that is substantially non-hybridizable to the polynucleotide and includes no more than one nucleotide from the 5xe2x80x2-end of the portion and (ii) a second fragment that is 3xe2x80x2 of the first fragment with reference to the intact oligonucleotide and is substantially hybridizable to the polynucleotide.
Another aspect of the present invention is a method for detecting a polynucleotide analyte. An oligonucleotide is reversibly hybridized with a polynucleotide analyte and a 5xe2x80x2-nuclease under isothermal conditions. The polynucleotide analyte serves as a recognition element to enable a 5xe2x80x2-nuclease to cleave the oligonucleotide to provide (i) a first fragment that is substantially non-hybridizable to the polynucleotide analyte and (ii) a second fragment that lies 3xe2x80x2 of the first fragment (in the intact oligonucleotide) and is substantially hybridizable to the polynucleotide analyte. At least a 100-fold molar excess of the first fragment and/or the second fragment are obtained relative to the molar amount of the polynucleotide analyte. The presence of the first fragment and/or the second fragment is detected, the presence thereof indicating the presence of the polynucleotide analyte.
Another embodiment of the present invention is a method for detecting a polynucleotide analyte. A combination is provided comprising a medium suspected of containing the polynucleotide analyte, an excess, relative to the suspected concentration of the polynucleotide analyte, of a first oligonucleotide at least a portion of which is capable of reversibly hybridizing with the polynucleotide analyte under isothermal conditions, a 5xe2x80x2-nuclease, and a second oligonucleotide having the characteristic of hybridizing to a site on the polynucleotide analyte that is 3xe2x80x2 of the site at which the first oligonucleotide hybridizes. The polynucleotide analyte is substantially fully hybridized to the second oligonucleotide under such isothermal conditions. The polynucleotide is reversibly hybridized under the isothermal conditions to the first oligonucleotide, which is cleaved as a function of the presence of the polynucleotide analyte to provide, in at least a 100-fold molar excess of the polynucleotide analyte, (i) a first fragment that is substantially non-hybridizable to the polynucleotide analyte and/or (ii) a second fragment that lies 3xe2x80x2 of the first fragment (in the intact first oligonucleotide) and is substantially hybridizable to the polynucleotide analyte. The presence of the first fragment and/or the second fragment is detected, the presence thereof indicating the presence of the polynucleotide analyte.
Another embodiment of the present invention is a method for detecting a DNA analyte. A combination is provided comprising a medium suspected of containing the DNA analyte, a first oligonucleotide at least a portion of which is capable of reversibly hybridizing with the DNA analyte under isothermal conditions, a 5xe2x80x2-nuclease, and a second oligonucleotide having the characteristic of hybridizing to a site on the DNA analyte that is 3xe2x80x2 of the site at which the first oligonucleotide hybridizes. The DNA analyte is substantially fully hybridized to the second oligonucleotide under isothermal conditions. The polynucleotide analyte is reversibly hybridized to the first oligonucleotide under isothermal conditions. The first oligonucleotide is cleaved to (i) a first fragment that is substantially non-hybridizable to the DNA analyte and (ii) a second fragment that lies 3xe2x80x2 of the first fragment (in the intact first oligonucleotide) and is substantially hybridizable to the DNA analyte. At least a 100-fold molar excess, relative to the DNA analyte, of the first fragment and/or the second fragment is produced. The presence of the first fragment and/or the second fragment is detected, the presence thereof indicating the presence of the DNA analyte.
Another embodiment of the present invention is a kit for detection of a polynucleotide. The kit comprises in packaged combination (a) a first oligonucleotide having the characteristic that, when reversibly hybridized under isothermal conditions to the polynucleotide, it is degraded by a 5xe2x80x2-nuclease to provide (i) a first fragment that is substantially non-hybridizable to the polynucleotide and (ii) a second fragment that is 3xe2x80x2 of the first fragment (in the first oligonucleotide) and is substantially hybridizable to the polynucleotide, (b) a second oligonucleotide having the characteristic of hybridizing to a site on the polynucleotide that is separated by no more than one nucleotide from the 3xe2x80x2-end of the site at which the first oligonucleotide hybridizes wherein the polynucleotide is substantially fully hybridized to the second oligonucleotide under the isothermal conditions, and (c) a 5xe2x80x2-nuclease.